Cell culture device and cell culture method

Abstract

To reconstruct cells into a desired morphology.SOLUTION: A cell culture device 1 includes a rotating vessel 20 that contains a liquid serving as a nutrient medium for cells C and rotates at 80 to 250 rpm, and a cell seeding member 30 that is fixed inside the rotating vessel 20 and has a seeding surface 30a on which the cells C are seeded. The rotating vessel 20 is hollow and has a generally cylindrical shape, has a bottom surface to which a rotating shaft 12 is connected, is arranged generally horizontally, and rotates in a circumferential direction. The rotating shaft 12 extends vertically or in a direction with an inclination of ±15° or less relative to the vertical direction and applies hydraulic pressure by the liquid to the cells C seeded in the cell seeding member 30 by the rotation of the rotating vessel 20.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to a cell culture device and a cell culture method. [Background technology]

[0002] Conventionally, autologous cartilage tissue transplantation for congenital or acquired tissue deformations in the face region, elbow, knee joints, etc., has problems such as the high invasiveness and quantitative limitations associated with cartilage harvesting. In recent years, there has been growing expectation for regenerative therapy using human chondrocytes as a treatment method, but to date it has not been possible to reconstruct cartilage tissue (cells) in a test tube that can be transplanted in one step as is. In addition, it is said that there are approximately 35 million patients with osteoarthritis of the knee in Japan alone.

[0003] For this reason, Patent Document 1 proposes a technique for reconstructing cartilage tissue, in which cells are suspended in a non-adherent state in a rotary culture vessel equipped with an agitator that rotates around a support pillar installed vertically from the bottom of the vessel body, and the cartilage-like tissue is cultured by efficiently bringing the cartilage-like tissue into contact with the bottom and inner wall of the vessel body by rotating the agitator, and applying mechanical stimulation, particularly shear force, to the surface of the cartilage-like tissue. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2020 / 130147 Summary of the Invention [Problem to be solved by the invention]

[0005] Here, in order to transplant the reconstructed cartilage tissue (cells) into a human, it is desirable that the reconstructed cartilage tissue (cells) have a form that corresponds to the part to which it is to be transplanted. However, the culture technology described in Patent Document 1 involves culturing the cells by applying stress to the cells floating inside the container body, which has the problem that it is difficult to reconstruct the cells into the desired morphology.

[0006] The present invention has been made to solve such problems, and has an object to provide a cell culture device and a cell culture method that enable cells to be reconstructed into a desired morphology. [Means for solving the problem]

[0007] (1) A cell culture device for culturing cells, comprising: A rotating vessel that contains a liquid serving as a nutrient medium for the cells and is rotated at 80 to 250 rpm; a cell seeding member that is fixed inside the rotating container and has a seeding surface on which the cells are seeded; A cell culture device, characterized in that, as the rotating container rotates, hydraulic pressure is applied by the liquid to the cells seeded on the cell seeding member.

[0008] According to the invention (1), a cell culture device for culturing cells includes a rotating container and a cell seeding member. The rotating vessel contains a liquid that serves as a nutrient medium for the cells and is rotated at 80 to 250 rpm. The cell seeding member is disposed inside the rotating vessel and has a seeding surface on which the cells are seeded. In the cell culture device, the rotating container rotates, so that a liquid pressure is applied to the cells seeded on the cell seeding member.

[0009] In the invention of (1), cells are seeded on the seeding surface of a cell seeding member fixed inside a rotating vessel, and the rotating vessel itself containing liquid inside is rotated at a relatively high speed of 80 to 250 rpm, thereby applying hydraulic pressure from the liquid to the cells seeded on the cell seeding member.

[0010] As a result, the seeding surface of the cell-seeding member is subjected to hydraulic pressure by the liquid, and by exposing the cells seeded on the seeding surface to the hydraulic pressure, the cells are reconstructed along the cell-seeding member, making it possible to reconstruct the cells into a form that corresponds to the shape of the cell-seeding member. Therefore, it is possible to provide a cell culture device that allows cells to be reconstructed into a desired morphology.

[0011] (2) The rotating vessel is hollow and cylindrical, has a rotating shaft connected to its bottom, is disposed substantially horizontally, and is rotated in a circumferential direction. The cell culture device according to (1), wherein the rotation axis extends in a vertical direction or in a direction inclined at an angle of ±15° or less with respect to the vertical direction.

[0012] This allows the rotating vessel, which is positioned approximately horizontally, to rotate in a circumferential direction, making it easier to control the movement of liquid inside the rotating vessel when rotating compared to when the rotation axis of the rotating vessel is horizontal, and making it easier to control the liquid pressure applied to the cells seeded on the cell seeding member.

[0013] (3) the cell-seeding member is formed in a plate shape having the seeding surface, The cell culture device according to (1) or (2), characterized in that the seeding surface is arranged facing the radial direction of the trajectory of the rotating container as it rotates or in a direction whose inclination with respect to the radial direction is within ±15°.

[0014] In this way, by forming the cell seeding component into a plate shape, and positioning the seeding surface formed into a flat shape so that the cells are seeded facing the radial direction of the trajectory of the rotating rotating container or in a direction with an inclination to the radial direction within ±15°, the entire seeding surface can be exposed to the liquid rotating in the rotational direction of the rotating container, making it possible to apply liquid pressure more efficiently to the cells seeded on the seeding surface.

[0015] (4) The cell-seeding member is formed in a box shape with the seeding surface being open, 3. The cell culture device according to claim 1, wherein the seeding surface is disposed facing a tangent to a trajectory of the rotating container as it rotates or in a direction whose inclination with respect to the tangent direction is within ±15°.

[0016] In the invention (4), the cell seeding member is formed into a box shape with an open seeding surface, and the seeding surface on which the cells are seeded is positioned facing the tangent direction of the trajectory of the rotating container or in a direction with an inclination to the tangent direction within ±15°. When the rotating vessel rotates, the liquid inside the rotating vessel is pulled by the wall in the tangential direction of the trajectory of the rotating vessel due to the interaction between the liquid and the wall of the rotating vessel, resulting in a flow of liquid inside the rotating vessel. In this state, the rotation speed of the wall and the cell seeding member fixed to the rotating vessel is faster than the rotation speed of the liquid near the outer periphery of the rotating vessel. This speed difference generates liquid pressure on the seeding surface of the cell seeding member. Since the cells are seeded on the seeding surface where such liquid pressure is generated, it becomes possible to apply liquid pressure to the cells more efficiently.

[0017] (5) A cell culture method for culturing cells, comprising the steps of: seeding the cells on a cell seeding member that is disposed inside a rotating container that contains a liquid serving as a nutrient medium for the cells, and has a seeding surface on which the cells are seeded; A cell culture method, comprising rotating the rotating vessel at 80 to 250 rpm and applying a liquid pressure from the liquid to the cells seeded on the cell seeding member.

[0018] The invention of (5) has the same effect as the invention of (1).

[0019] (6) seeding the cells in the cell-seeding member and leaving the cell-seeding member for a first predetermined period of time; a first rotation step of rotating the rotating container at a first rotation speed for a second predetermined period after the standing step; The cell culture method according to (5), further comprising: a second rotation step of rotating the rotating vessel at a second rotation speed, which is faster than the first rotation speed, for a third predetermined period after the first rotation step.

[0020] In the sixth aspect of the present invention, the cell culture method includes a stationary step, a first rotation step, and a second rotation step. The leaving step includes seeding cells in the cell-seeding member and leaving the cell-seeding member for a first predetermined period. In the first rotation step, after the standing step, the rotating vessel is rotated at the first rotation speed for a second predetermined period of time. In the second rotating step, the rotating vessel is rotated at a second rotation speed, which is faster than the first rotation speed, for a third predetermined period after the first rotation step.

[0021] According to the invention (6), a stationary step allows the cells seeded on the cell-seeding member to settle on the cell-seeding member to the extent that they will not flow away even if the cell-seeding member is moved, and then a first rotation step applies liquid pressure to the cells to reconstruct the cells and stabilize the cells on the cell-seeding member, and then a second rotation step rotates the rotating container at a rotation speed faster than that of the first rotation step, thereby promoting cell reconstruction.

[0022] This allows the step of applying fluid pressure to the cells to be divided into multiple stages, and the fluid pressure can be gradually increased, making it possible to steadily promote cell reconstruction. Effect of the Invention

[0023] According to the present invention, it is possible to provide a cell culture device and a cell culture method that enable cells to be reconstructed into a desired morphology. [Brief description of the drawings]

[0024] [Figure 1] FIG. 1 is a diagram illustrating a cell culture device according to an embodiment of the present invention. [Diagram 2] 1 is a diagram illustrating a rotating container according to an embodiment of the present invention. FIG. [Diagram 3] 3A and 3B are diagrams illustrating an example of a cell-seeding member according to an embodiment of the present invention, in which Fig. 3(a) shows an example in which the cell-seeding member is formed in a plate shape, and Fig. 3(b) shows an example in which the cell-seeding member is formed in a box shape with an open seeding surface. [Figure 4] 1A to 1C are diagrams illustrating examples of arrangement of a cell-seeding member inside a rotating container according to an embodiment of the present invention. [Diagram 5] 1A to 1C are diagrams illustrating examples of arrangement of a cell-seeding member inside a rotating container according to an embodiment of the present invention. [Figure 6] 1A to 1C are diagrams illustrating examples of arrangement of a cell-seeding member inside a rotating container according to an embodiment of the present invention. [Figure 7] 1A to 1C are diagrams illustrating examples of arrangement of a cell-seeding member inside a rotating container according to an embodiment of the present invention. [Figure 8] 1A to 1C are diagrams illustrating examples of arrangement of a cell-seeding member inside a rotating container according to an embodiment of the present invention. [Figure 9] 9A and 9B are images of elastic cartilage tissue reconstructed from auricular elastic chondrocytes by the cell culture method according to an embodiment of the present invention. Fig. 9(a) is an image showing the entire reconstructed elastic cartilage tissue. Fig. 9(b) is an image of the Alcian blue-stained positive image of the elastic cartilage tissue shown in Fig. 9(a) enlarged 100 times. [Figure 10] 10 shows images of a reconstructed articular cartilage (hyaline cartilage) film by a cell culture method according to an embodiment of the present invention. Fig. 10(a) is an image showing the entire reconstructed articular cartilage (hyaline cartilage) film. Fig. 10(b) is an image of the Alcian blue-stained positive image of the articular cartilage (hyaline cartilage) film shown in Fig. 10(a) enlarged by 40 times. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] [Outline of cell culture equipment and cell culture methods] The cell culture device and cell culture method according to the embodiment of the present invention make it possible to reconstruct various forms from cultured chondrocytes without using a scaffold material, such as cartilage tissue (5 mm thick) applicable to rhinoplasty and cartilage film (2 mm thick) applicable to osteoarthritis of the knee.

[0026] In order for cells (such as cartilage tissue) to mature, they need loads and stresses such as stimulation, low oxygen concentration, and high carbon dioxide concentration. The cell culture device and cell culture method according to this embodiment are a technology for regenerating cells by applying a load of fluid pressure (dynamic pressure caused by liquid) to the cells by rotating the liquid nutrient medium for the cells at high speed.

[0027] The cell culture apparatus and cell culture method according to the present embodiment are suitable for reconstructing cartilage tissue derived from human chondrocyte precursor cells or mature cells, but are not limited thereto and can also be applied to rhinoplasty, ear reconstruction, and osteoarthritis of the knee.

[0028] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, modes for carrying out the present invention (hereinafter, referred to as embodiments) will be described in detail with reference to the accompanying drawings.

[0029] [Cell culture device configuration] 1 is a diagram illustrating a cell culture device according to an embodiment of the present invention. Note that, throughout the description of the embodiment, the same or similar elements are given the same numbers or symbols, and their description will be omitted or simplified.

[0030] The cell culture device 1 comprises a base 5, a rotating device 10 installed on the base 5, a rotating container 20 connected to the rotating device 10, and a cell seeding member 30 (see Figure 2 described later) placed in the rotating container 20, and cultures cells.

[0031] In this embodiment, the "cells" include culturable cells such as chondrocytes, perichondrocytes, osteocytes, osteoblasts, periosteal cells, tendon cells, epidermal cells, fibroblasts, mucosal cells, cardiac muscle cells, and peritoneal cells.

[0032] The base 5 is installed on a horizontal plane and includes a base 5a having a mounting surface on which the rotating device 10 is placed, and a support portion 5b that stands upward from the base 5a and rotatably supports the upper end side of the rotating shaft that rotates the rotating container 20, and fixes the rotating device 10.

[0033] <Rotation device 10> The rotating device 10 includes a main body 11, a rotating shaft 12, a connection adapter 13, and a shaft adapter 14, and rotates a rotating container 20 connected thereto.

[0034] The main body 11 is equipped with a motor (not shown) that rotates the rotating shaft 12 using an externally supplied power source, and a control unit (not shown) that controls the rotation of the motor, and rotates the rotating shaft 12 at a rotation speed set in the range of 80 to 250 rpm under the control of the control unit based on settings made by an operator.

[0035] The rotating shaft 12 is a shaft member that extends in the vertical direction, with its upper side protruding above the main body 11, and is rotated in the circumferential direction around its axis by the motor of the main body 11.

[0036] The connection adapter 13 has its center located at the position where the axis of the rotating shaft 12 extends, and includes a connection part 13a and a mounting part 13b.

[0037] The connecting portion 13 a is formed in a shaft shape, and the lower end side is detachably fixed to the upper end of the rotating shaft 12 or the upper end of the connecting shaft 23 of the rotating container 20 . The mounting portion 13b is formed in a disk shape, the center of which is fixed to the upper end of the connecting portion 13a, the rotating container 20 is detachably fixed thereto, and the center of the rotating container 20 is disposed at a position where the axis of the rotating shaft 12 extends.

[0038] The shaft adapter 14 is formed in a shaft shape, and its lower end side is detachably fixed to the upper end of the connecting shaft 23 of the rotating container 20 installed in the uppermost stage. In addition, the upper end side of the shaft adapter 14 is rotatably held at the upper end of the support part 5b of the base 5.

[0039] Here, the support portion 5b may be provided with a horizontal member 5b1 that protrudes horizontally at the upper end side and has a U-shaped notch formed on its side edge, and an engagement member 5b2 that detachably engages with the horizontal member 5b1 and has a U-shaped notch formed on its side edge. The support portion 5b forms a holding hole that rotatably holds the shaft adapter 14 or the upper end side of the connection shaft 23 of the rotating container 20 by engaging the horizontal member 5b1 and the engagement member 5b2 so that their notches face each other.

[0040] As a result, the shaft that rotates together with the rotating vessel 20 (e.g., the rotating shaft 12, the connection part 13a, and the shaft adapter 14) can be held at the top and bottom in the center of the rotating vessel 20, preventing the rotating vessel 20 from wobbling during rotation. Furthermore, by forming a holding hole that rotatably holds the shaft adapter 14 and the upper end side of the connecting shaft 23 of the rotating vessel 20 using the horizontal member 5b1 and the engaging member 5b2 that detachably engages with this horizontal member 5b1, it becomes possible to set the rotating vessel 20, the shaft adapter 14, etc. on the rotating device 10 with the engaging member 5b2 removed, and then engage the engaging member 5b2 with the horizontal member 5b1. As a result, it becomes easy to set the rotating vessel 20 on the rotating device 10 and to stabilize the rotation of the rotating vessel 20.

[0041] In addition, the rotating device 10 may be provided with a leakage prevention cover 15 that is formed in a plate shape and is arranged on the upper surface of the main body 11 around the rotating shaft 12 that protrudes from the upper surface of the main body 11, covering the portion of the main body 11 where the rotating shaft 12 protrudes, and prevents liquid from entering the inside of the main body 11 from that portion.

[0042] In addition, the rotating device 10 may be provided with a dust / dirt guard 16 which is formed in a plate shape, is disposed between the connecting shaft 23 of the rotating container 20 installed at the top stage and the shaft adapter 14, covers the upper part of the rotating container 20, and prevents dust and dirt from adhering to the rotating container 20.

[0043] <Rotating Container 20> Fig. 2 is a diagram illustrating a rotating container according to an embodiment of the present invention, as viewed from the side. The rotating container 20 has a substantially circular shape in a plan view, is formed into a hollow, substantially cylindrical shape, and includes a first member 21 placed on the upper surface of the mounting portion 13b of the connection adapter 13, a second member 22 placed on the first member 21 and connected to the first member 21, a connection shaft 23 extending upward from the upper surface of the second member 22, and a lid 24 engaging with the second member 22. The rotating container 20 is, for example, a vessel.

[0044] The rotating container 20 is placed approximately horizontally (horizontally or at an inclination of within ±15° relative to the horizontal) on the main body 11, and the driving force of the motor of the main body 11 is transmitted via the rotating shaft 12 and the connection adapter 13, causing the rotating container 20 to rotate in a circumferential direction about a rotating axis RS that passes through the center of the rotating container 20 in a plan view and extends in the vertical direction. It is desirable that the rotating axis RS extends in the vertical direction or in a direction at an inclination of within ±15° relative to the vertical direction.

[0045] The first member 21 has a bottom plate and a side wall extending upward from the outer edge of the bottom plate, is open at the top, and the bottom plate is detachably fixed to the upper surface of the mounting part 13b of the connection adaptor 13. The first member 21 also has an oxygen-permeable membrane for taking in external oxygen (air) that is essential for cellular metabolism.

[0046] The second member 22 has a top plate and a side wall extending downward from the outer edge of the top plate, is open at the bottom, and the lower end side of the side wall is detachably fixed to the upper end side of the first member 21, resulting in a liquid-tight state. By connecting the first member 21 and the second member 22, a cell culture space S for culturing cells is formed in the rotating container 20.

[0047] A nutrient medium liquid for the cells is contained in the cell culture space S. Examples of the nutrient medium liquid include basic fibroblast growth factor, insulin-like growth factor 1, transforming growth factor β1, β2, β3, bone morphogenetic protein, platelet-rich plasma, and the like.

[0048] Further, a through hole 22a is formed in the top plate of the second member 22. Further, an access portion 22b and a connection shaft 23 are provided in the top plate of the second member 22.

[0049] The access portion 22b extends upward along the outer edge of the through hole 22a and is formed in a cylindrical shape that is open at the top and bottom. It is preferable that a pair of access portions 22b is provided at positions that are line-symmetrical with respect to the rotation axis RS. This improves the rotation balance of the rotating container 20.

[0050] The connection shaft 23 is formed in a cylindrical shape extending upward from the upper surface of the top plate of the second member 22, and its center in a top view is disposed at the center of the top plate of the second member 22 (the position where the rotation axis RS extends).

[0051] The lid 24 is disposed on the upper end side of the access portion 22b and is removably fixed to the access portion 22b, closing the upper end side of the access portion 22b and providing a liquid-tight state.

[0052] <Cell seeding member 30> Fig. 3 is a diagram illustrating an example of a cell-seeding member according to an embodiment of the present invention, in which Fig. 3(a) shows an example in which the cell-seeding member is formed into a plate shape, and Fig. 3(b) shows an example in which the cell-seeding member is formed into a box shape with an open seeding surface.

[0053] The cell-seeding member 30 is fixed inside the rotating container 20 and has a seeding surface 30a on which cells C are seeded. The cell-seeding member 30 is not limited to the example shown in Fig. 3, and may have any shape suitable for the application of the cultured cells, such as an uneven shape, a wavy shape, or a shape imitating the outer shape of any part of a bone.

[0054] 4 to 8 are diagrams illustrating examples of arrangement of the cell-seeding member inside the rotating container according to the embodiment of the present invention. Figures 4 to 8 are diagrams showing the inside of the rotating container from above, and in order to make it easier to understand the position of the cell-seeding member, the access part 22b of the second member 22 and the connection shaft 23 are indicated by two-dot chain lines.

[0055] In the example shown in Fig. 3(a), the cell-seeding member 30A is formed in a plate shape having a seeding surface 30a. In this example, as shown in Fig. 4, the seeding surface 30a on which the cells C are seeded faces the rotation axis RS of the rotating vessel 20, and the cell-seeding member 30A is fixed to the rotating vessel 20 at a position furthest from the rotation axis RS (a position where the cell-seeding member 30A abuts against the side wall of the rotating vessel 20) in the cell culture space S formed inside the rotating vessel 20. As a result, the seeded cells C can be exposed to the seeding surface 30a at a position where the flow rate of the liquid flowing in the cell culture space S is relatively fast due to the rotation of the rotating vessel 20, and therefore a higher load can be applied to the cells C.

[0056] 3(a), the cell-seeding member 30A is a plate-like body having a width of 18 mm, a height of 10.5 mm, and a thickness of 1 mm. The dimensions of the cell-seeding member 30A can be arbitrarily set depending on the cells to be cultured and the size of the cell culture space S of the rotating container 20 to be housed therein.

[0057] In the example shown in FIG. 4, the seeding surface 30a is disposed facing the radial direction R of the trajectory of the rotating rotary vessel 20 or a direction whose inclination with respect to the radial direction R is within ±15°.

[0058] In the example shown in Fig. 3(b), the cell-seeding member 30B is formed in a box shape with the seeding surface 30a open. In this example, as shown in Fig. 5 to Fig. 8, the cell-seeding member 30B is fixed to the rotating vessel 20 in the cell culture space S formed inside the rotating vessel 20, below the access part 22b, with the seeding surface 30a on which the cells C are seeded facing the rotation direction dr of the rotating vessel 20.

[0059] 3(b), the cell-seeding member 30B is formed in a box shape with a width of 18 mm, a height of 10.5 mm, and a thickness of 6 mm, and a notch with an inner shape of width of 12.4 mm, a height of 7.2 mm, and a thickness of 5 mm formed on the inside of the outer shape of a substantially rectangular parallelepiped. The dimensions of the cell-seeding member 30B can be set to any dimensions depending on the cells to be cultured and the size of the cell culture space S of the rotating vessel 20 to be accommodated.

[0060] In the example shown in FIG. 5, the seeding surface 30a is disposed facing the rotation direction dr of the rotating vessel 20, and facing the tangential direction td of the trajectory of the rotating vessel 20.

[0061] In the example shown in Figure 3(b), the seeding surface 30a may be arranged facing the rotation direction dr of the rotating vessel 20, and may be arranged facing a direction whose inclination with respect to the tangential direction td of the trajectory of the rotating vessel 20 is within ±15°.

[0062] For example, in the example shown in Figure 6, the sowing surface 30a is arranged facing the rotational direction dr of the rotating vessel 20, compared to the example shown in Figure 5, and is arranged facing a direction inclined 15° to the outside of the rotating vessel 20 with respect to the tangential direction td of the trajectory of the rotating rotating vessel 20.

[0063] In addition, in the example shown in Figure 7, the sowing surface 30a, compared to the example shown in Figure 5, is arranged facing the rotational direction dr of the rotating vessel 20, and is arranged facing a direction inclined by 15° inwardly of the rotating vessel 20 with respect to the tangential direction td of the trajectory of the rotating rotating vessel 20.

[0064] The direction in which the seeding surface 30a faces is not limited to the examples shown in Figures 4 to 7, and may be arranged at any angle with respect to the radial direction R of the trajectory of the rotating vessel 20 or the tangential direction td of the trajectory of the rotating vessel 20.

[0065] Furthermore, a plurality of cell-seeding members 30 may be disposed in one cell culture space S. For example, in the example shown in Figure 8, two cell seeding members 30B are arranged in the cell culture space S, point-symmetrically with respect to the position where the rotation axis RS extends (the center of the rotating vessel 20 in a planar view), and each is fixed to the rotating vessel 20 below the access portion 22b, with the seeding surface 30a facing toward the rotation direction dr of the rotating vessel 20.

[0066] [Cell culture method] Next, a cell culture method will be described. The cell culture device 1 according to this embodiment is suitable for carrying out the cell culture method according to this embodiment, and in the following description, a cell culture method using the cell culture device 1 will be described. However, the cell culture method according to the present invention may use other devices instead of the cell culture device 1.

[0067] In the cell culture method according to this embodiment, for example, as shown in FIG. 4, cells C are seeded onto a cell seeding member 30 that is placed inside a rotating container 20 (cell culture space S) that contains a liquid serving as a nutrient medium for the cells and has a seeding surface 30a on which the cells C are seeded, the rotating container 20 is rotated at 80 to 250 rpm, and hydraulic pressure from the liquid is applied to the cells C seeded onto the cell seeding member 30.

[0068] In detail, the cell culture method includes a stationary step, a first rotation step performed after the stationary step, and a second rotation step performed after the first rotation step.

[0069] In the leaving step, the cells C are seeded on the cell-seeding member 30 and left to stand for a first predetermined period (e.g., 3 to 7 days). In the leaving step, it is desirable to arrange the seeding surface 30a of the cell-seeding member 30 horizontally. This makes it possible to prevent the cells C seeded on the seeding surface 30a from flowing off the seeding surface 30a before they are established. Note that the cells C may be seeded after applying artificial collagen to the seeding surface 30a. This makes it difficult for the cells C seeded on the seeding surface 30a to flow away.

[0070] In the first rotation step, the rotating vessel 20 is rotated at a first rotation speed (20 to 30 rpm) for a second predetermined period (e.g., within one day) after the stationary step. In detail, the rotating vessel 20, in which the cell-seeding member 30 having the cells C fixed on the seeding surface 30a is placed in the cell culture space S, is set in the rotating device 10 (see FIG. 1), and the rotating shaft 12 (see FIG. 1) is rotated under the control of the control unit of the main body 11 (see FIG. 1), thereby rotating the rotating vessel 20 at the first rotation speed (e.g., 20 to 30 rpm).

[0071] In the second rotation step, the rotating vessel 20 is rotated at a second rotation speed (preferably 100 to 200 rpm) that is faster than the first rotation speed for a third predetermined period (e.g., 1 to 2 weeks) after the first rotation step. In detail, the rotating vessel 20 is rotated at the second rotation speed (preferably 100 to 200 rpm) from the state of the first rotation step by rotating the rotating shaft 12 (see FIG. 1) under the control of the control unit of the main body 11 (see FIG. 1).

[0072] FIG. 9 is an image of elastic cartilage tissue reconstructed from auricular elastic chondrocytes by the cell culture method according to an embodiment of the present invention. Fig. 9(a) is an image showing the entire reconstructed elastic cartilage tissue. The scale shown at the bottom of Fig. 9(a) indicates 1 mm per scale. Figure 9(b) is a 100x magnified image of the Alcian blue positive staining of the elastic cartilage tissue shown in Figure 9(a). The color in Figure 9(b) is displayed in grayscale with Alcian blue. do.

[0073] By using the cell culture method of this embodiment, elastic cartilage tissue with strength equivalent to that of human cartilage tissue was reconstructed from ear elastic chondrocytes with almost 100% reproducibility without using a scaffold material. Not only was this tissue stained with Alcian blue, which indicates cartilage tissue, to the same extent as human cartilage tissue, but central necrosis was not observed (see FIG. 9).

[0074] Conventionally, cultured cartilage tissue shrinks after being transplanted into an animal or the like, and it has been impossible to control the size before transplantation. According to the cell culture method of this embodiment, not only can a cartilage matrix be produced during rotation of the rotating container 20, but also the contraction phenomenon that occurs during implantation in a living body can be reproduced. For example, the size of the cartilage tissue can be controlled by adding cells C to the contracted cartilage tissue during rotation. The additional administration of chondrocytes is considered to be the main reason why "the reconstructed cartilage has mechanical strength, produces matrix similar to that of cartilage tissue in a living body, and does not show central necrosis."

[0075] Furthermore, according to the cell culture method of this embodiment, by adjusting the size and shape of the rotating container 20 and the cell seeding member 30, it is also possible to reconstruct cartilage tissue of approximately 40 mm x 10 mm x 10 mm, which is sufficient for rhinoplasty.

[0076] Furthermore, using the cell culture method of this embodiment, we succeeded in reconstructing articular cartilage (hyaline cartilage) films measuring 50 mm x 10 mm x 2 mm that are applicable to osteoarthritis of the knee from human articular cartilage (hyaline cartilage) with almost 100% reproducibility without using a scaffold material.

[0077] FIG. 10 is an image of a film of reconstructed articular cartilage (hyaline cartilage) by a cell culture method according to an embodiment of the present invention. Fig. 10(a) is an image showing the entire reconstructed articular cartilage (hyaline cartilage) film. The scale indicated at the bottom of Fig. 10(a) is 1 mm per scale. Figure 10(b) is a 40x magnification image of the Alcian blue positive staining of the articular cartilage (hyaline cartilage) film shown in Figure 10(a). The color in Figure 10(b) is displayed in grayscale with Alcian blue.

[0078] The articular cartilage (hyaline cartilage) film reconstructed by the cell culture method of the present embodiment has excellent elasticity and flexibility, and can therefore be used to cover and suture a knee joint. In addition, the articular cartilage (hyaline cartilage) film can be stored at 4°C, making it easy to transport. In addition, the articular cartilage (hyaline cartilage) film can also be reconstructed to a size of approximately 100 mm x 100 mm x 5 mm by adjusting the size and shape of the rotating container 20 and the cell seeding member 30, just like the elastic cartilage tissue. Thus, the cell culture method of the present embodiment is a cell manipulation technique for reconstructing histologically mature cultured cartilage tissue with mechanical strength suitable for clinical application from human cartilage precursor / mature chondrocytes. In addition, the cells reconstructed by the cell culture method of the present embodiment have mechanical strength almost equivalent to that of cartilage tissue in vivo without using scaffold materials such as artificial substances, and therefore are easy to handle.

[0079] As described above, according to this embodiment, the seeding surface 30a of the cell seeding member 30 receives the liquid pressure, and the cells C seeded on the seeding surface 30a are exposed to the liquid pressure, thereby causing the cells C to be reconstructed along the cell seeding member 30, and the cells C can be reconstructed into a form that corresponds to the shape of the cell seeding member 30. Therefore, it is possible to provide a cell culture device that allows cells to be reconstructed into a desired morphology.

[0080] Furthermore, according to this embodiment, the rotating vessel 20 arranged approximately horizontally can be rotated in a circumferential direction. Therefore, when the rotating vessel 20 is rotated, it is easier to control the movement of liquid inside the rotating vessel 20 compared to when the rotation axis of the rotating vessel 20 is horizontal, and it is easier to control the liquid pressure applied to the cells C seeded on the cell seeding member 30.

[0081] Furthermore, according to this embodiment, the cell seeding member 30A is formed in a plate shape, and the cells C are seeded facing the radial direction R of the trajectory of the rotating rotating vessel 20 or in a direction with an inclination relative to the radial direction R within ±15°. By arranging the seeding surface 30a formed in a planar shape, the entire seeding surface 30a can be exposed to the liquid rotating in the rotational direction of the rotating vessel 20, thereby making it possible to apply liquid pressure more efficiently to the cells seeded on the seeding surface 30a.

[0082] Furthermore, according to this embodiment, the cell seeding member 30B is formed into a box shape with an open seeding surface 30a, and the seeding surface 30a on which the cells C are seeded is positioned facing the tangential direction td of the trajectory of the rotating rotating container 20 or in a direction with an inclination relative to the tangential direction td within ±15°. When the rotating vessel 20 rotates, the liquid inside the rotating vessel 20 is pulled by the wall surface in the tangential direction td of the trajectory of the rotating vessel 20 due to the interaction between the liquid and the wall surface of the rotating vessel 20, resulting in a flow of liquid inside the rotating vessel 20. In this state, the rotation speed of the wall surface and the cell-seeding member 30B fixed to the rotating vessel 20 is faster than the rotation speed of the liquid near the outer periphery of the rotating vessel 20. This speed difference generates liquid pressure on the seeding surface 30a of the cell-seeding member 30B. Since the cells C are seeded on the seeding surface 30a where such a liquid pressure is generated, it becomes possible to apply liquid pressure to the cells more efficiently.

[0083] Furthermore, according to this embodiment, the stationary step allows the cells C seeded on the cell seeding member 30 to settle on the cell seeding member 30 to the extent that they will not flow away even if the cell seeding member 30 is moved, and then, in the first rotation step, liquid pressure is applied to the cells C to reconstruct the cells C and stabilize the cells C on the cell seeding member 30. Thereafter, in the second rotation step, the rotating container 20 is rotated at a rotation speed faster than that in the first rotation step, thereby promoting the reconstruction of the cells C.

[0084] This allows the step of applying fluid pressure to the cells to be divided into multiple stages, and the fluid pressure can be gradually increased, making it possible to steadily promote cell reconstruction.

[0085] Although the embodiment of the present invention has been described above, it goes without saying that the technical scope of the present invention is not limited to the contents of the above embodiment. It is clear to those skilled in the art that various modifications and improvements can be made to the above embodiment. Furthermore, it is clear from the description of the claims that forms with such modifications or improvements are also included in the technical scope of the present invention. [Explanation of symbols]

[0086] C cell R Radial direction RS Rotating Axis S Cell culture space dr Rotation direction td tangential direction 1 Cell culture device 5. Bass 5a Foundation 5b Support part 5b1 Horizontal members 5b2 Engagement member 10 Rotating device 11 Main unit 12 Rotation axis 13 Connection adapter 13a Connection 13b Placement part 14 Axis Adapter 15 Leak prevention cover 16 Dust and dirt prevention 20 Rotating Container 21 First member 22 Second member 22a Through hole 22b Access section 23 Connecting shaft 24 Lid 30, 30A, 30B Cell seeding member 30a Sowing surface

Claims

1. A cell culture device for culturing cells, comprising: A rotating vessel that contains a liquid serving as a nutrient medium for the cells and is rotated at 80 to 250 rpm; a cell seeding member that is fixed inside the rotating container and has a seeding surface on which the cells are seeded; A cell culture device, characterized in that, as the rotating container rotates, hydraulic pressure is applied by the liquid to the cells seeded on the cell seeding member.

2. The rotating container is hollow and generally cylindrical, has a rotating shaft connected to its bottom surface, is disposed generally horizontally, and is rotated in a circumferential direction.

2. The cell culture device according to claim 1, wherein the rotation axis extends in a vertical direction or in a direction tilted at an angle of ±15° or less with respect to the vertical direction.

3. The cell-seeding member is formed in a plate shape having the seeding surface, 3. The cell culture device according to claim 1, wherein the seeding surface is disposed facing a radial direction of a trajectory of the rotating container as it rotates or in a direction whose inclination with respect to the radial direction is within ±15°.

4. The cell seeding member is formed in a box shape with the seeding surface being open, 3. The cell culture device according to claim 1, wherein the seeding surface is disposed facing a tangent to a trajectory of the rotating container as it rotates or in a direction inclined at an angle of ±15° or less with respect to the tangent.

5. A cell culture method for culturing cells, comprising the steps of: seeding the cells on a cell seeding member that is disposed inside a rotating container that contains a liquid serving as a nutrient medium for the cells, and has a seeding surface on which the cells are seeded; A cell culture method comprising rotating the rotating vessel at 80 to 250 rpm and applying a liquid pressure from the liquid to the cells seeded on the cell seeding member.

6. a step of seeding the cells in the cell seeding member and leaving the cell seeding member for a first predetermined period of time; a first rotation step of rotating the rotating container at a first rotation speed for a second predetermined period after the standing step; 6. The cell culture method according to claim 5, further comprising: a second rotation step of rotating the rotating vessel at a second rotation speed, which is faster than the first rotation speed, for a third predetermined period after the first rotation step.

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